Heterologous expression and enzymatic characteristics of sulfatase from Lactobacillus plantarum dy-1.

Barley, rich in bioactive components including dietary fiber, polyphenolic compounds and functional proteins, exhibits health benefits such as regulating glucose and lipid metabolism. Previous studies have found that the content and composition of free phenolic acids in barley may be significantly changed by fermentation with the laboratory patented strain Lactobacillus plantarum dy-1 (L. p dy-1), but the mechanism of enzymatic release of phenolic acid remains to be elucidated. Based on this, this study aimed to identify the key enzyme in L. p dy-1 responsible for releasing the bound phenolic acid and to further analyze its enzymatic properties. The Carbohydrate-Active enZYmes database revealed that L. p dy-1 encodes 7 types of auxiliary enzymes, among which we have identified a membrane sulfatase. The enzyme gene LPMS05445 was heterologous to that expressed in E. coli, and a recombinant strain was induced to produce the target protein and purified. The molecular weight of the purified enzyme was about 59.9 kDa, with 578.21 U mg-1 enzyme activity. The optimal temperature and pH for LPMS05445 expression were 40 °C and 7.0, respectively. Furthermore, enzymatic hydrolysis by LPMS05445 can obviously change the surface microstructure of dietary fiber from barley bran and enhance the release of bound phenolic acid, thereby increasing the free phenolic acid content and improving its physiological function. In conclusion, sulfatase produced by Lactobacillus plantarum dy-1 plays a key role in releasing bound phenolic acids during the fermentation of barley.

Preparation and Application of an Inexpensive α-Formylglycine Building Block Compatible with Fmoc Solid-Phase Peptide Synthesis.

α-Formylglycine (fGly) is a rare residue located in the active site of sulfatases and serves as a precursor to pharmaceutically relevant motifs. The installation of fGly motifs into peptides is currently challenging due to degradation under the acidic and nucleophile-rich conditions accompanying resin cleavage during solid-phase peptide synthesis. We report the synthesis of acid- and nucleophile-tolerant α-formylglycine building blocks from vitamin C and use them to prepare callyaerin A, a macrocyclic peptide containing an fGly-derived motif.

SulfAtlas, the sulfatase database: state of the art and new developments.

SulfAtlas (https://sulfatlas.sb-roscoff.fr/) is a knowledge-based resource dedicated to a sequence-based classification of sulfatases. Currently four sulfatase families exist (S1-S4) and the largest family (S1, formylglycine-dependent sulfatases) is divided into subfamilies by a phylogenetic approach, each subfamily corresponding to either a single characterized specificity (or few specificities in some cases) or to unknown substrates. Sequences are linked to their biochemical and structural information according to an expert scrutiny of the available literature. Database browsing was initially made possible both through a keyword search engine and a specific sequence similarity (BLAST) server. In this article, we will briefly summarize the experimental progresses in the sulfatase field in the last 6 years. To improve and speed up the (sub)family assignment of sulfatases in (meta)genomic data, we have developed a new, freely-accessible search engine using Hidden Markov model (HMM) for each (sub)family. This new tool (SulfAtlas HMM) is also a key part of the internal pipeline used to regularly update the database. SulfAtlas resource has indeed significantly grown since its creation in 2016, from 4550 sequences to 162 430 sequences in August 2022.

Engineering Pseudomonas aeruginosa arylsulfatase for hydrolysis of α-configured steroid sulfates.

Steroid sulfate esters are important metabolites for anti-doping efforts in sports, pathology and research. Analysis of these metabolites is facilitated by hydrolysis using either acid or enzymatic catalysis. Although enzymatic hydrolysis is preferred for operating at neutral pH, no known enzyme is capable of hydrolyzing all steroid sulfate metabolites. Pseudomonas aeruginosa arylsulfatase (PaS) is ideal for the hydrolysis of ß-configured steroid sulfates but like other known class I sulfatases it is inefficient at hydrolyzing α-configured steroid sulfates. We have used directed evolution with liquid chromatography mass spectrometry screening to find variants capable of hydrolyzing a α-configured steroid sulfate: etiocholanolone sulfate (ECS). After targeting two regions of PaS, four residues were identified and optimized to yield a final variant with a total of seven mutations (DRN-PaS) capable of hydrolyzing ECS ~80 times faster than the best PaS variant previously available. This DRN-PaS also shows improved activity for other α-configured steroid sulfates. Simultaneous mutagenesis was essential to obtain DRN-PaS due to complementarity between targeted residues.

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota.

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.

Structural insights into choline-O-sulfatase reveal the molecular determinants for ligand binding.

Choline-O-sulfatase (COSe; EC 3.1.6.6) is a member of the alkaline phosphatase (AP) superfamily, and its natural function is to hydrolyze choline-O-sulfate into choline and sulfate. Despite its natural function, the major interest in this enzyme resides in the landmark catalytic/substrate promiscuity of sulfatases, which has led to attention in the biotechnological field due to their potential in protein engineering. In this work, an in-depth structural analysis of wild-type Sinorhizobium (Ensifer) meliloti COSe (SmeCOSe) and its C54S active-site mutant is reported. The binding mode of this AP superfamily member to both products of the reaction (sulfate and choline) and to a substrate-like compound are shown for the first time. The structures further confirm the importance of the C-terminal extension of the enzyme in becoming part of the active site and participating in enzyme activity through dynamic intra-subunit and inter-subunit hydrogen bonds (Asn146A-Asp500B-Asn498B). These residues act as the `gatekeeper' responsible for the open/closed conformations of the enzyme, in addition to assisting in ligand binding through the rearrangement of Leu499 (with a movement of approximately 5 Å). Trp129 and His145 clamp the quaternary ammonium moiety of choline and also connect the catalytic cleft to the C-terminus of an adjacent protomer. The structural information reported here contrasts with the proposed role of conformational dynamics in promoting the enzymatic catalytic proficiency of an enzyme.

Structural analysis of the sulfatase AmAS from Akkermansia muciniphila.

Akkermansia muciniphila, an anaerobic Gram-negative bacterium, is a major intestinal commensal bacterium that can modulate the host immune response. It colonizes the mucosal layer and produces nutrients for the gut mucosa and other commensal bacteria. It is believed that mucin desulfation is the rate-limiting step in the mucin-degradation process, and bacterial sulfatases that carry out mucin desulfation have been well studied. However, little is known about the structural characteristics of A. muciniphila sulfatases. Here, the crystal structure of the premature form of the A. muciniphila sulfatase AmAS was determined. Structural analysis combined with docking experiments defined the critical active-site residues that are responsible for catalysis. The loop regions I-V were proposed to be essential for substrate binding. Structure-based sequence alignment and structural superposition allow further elucidation of how different subclasses of formylglycine-dependent sulfatases (FGly sulfatases) adopt the same catalytic mechanism but exhibit diverse substrate specificities. These results advance the understanding of the substrate-recognition mechanisms of A. muciniphila FGly-type sulfatases. Structural variations around the active sites account for the different substrate-binding properties. These results will enhance the understanding of the roles of bacterial sulfatases in the metabolism of glycans and host-microbe interactions in the human gut environment.

A single sulfatase is required to access colonic mucin by a gut bacterium.

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.

A novel thermostable prokaryotic fucoidan active sulfatase PsFucS1 with an unusual quaternary hexameric structure.

Fucoidans are sulfated, fucose-rich marine polysaccharides primarily found in cell walls of brown seaweeds (macroalgae). Fucoidans are known to possess beneficial bioactivities depending on their structure and sulfation degree. Here, we report the first functional characterization and the first crystal structure of a prokaryotic sulfatase, PsFucS1, belonging to sulfatase subfamily S1_13, able to release sulfate from fucoidan oligosaccharides. PsFucS1 was identified in the genome of a Pseudoalteromonas sp. isolated from sea cucumber gut. PsFucS1 (57 kDa) is Ca2+ dependent and has an unusually high optimal temperature (68 °C) and thermostability. Further, the PsFucS1 displays a unique quaternary hexameric structure comprising a tight trimeric dimer complex. The structural data imply that this hexamer formation results from an uncommon interaction of each PsFucS1 monomer that is oriented perpendicular to the common dimer interface (~ 1500 Å2) that can be found in analogous sulfatases. The uncommon interaction involves interfacing (1246 Å2) through a bundle of α-helices in the N-terminal domain to form a trimeric ring structure. The high thermostability may be related to this unusual quaternary hexameric structure formation that is suggested to represent a novel protein thermostabilization mechanism.

Genomic and in silico protein structural analyses provide insights into marine polysaccharide-degrading enzymes in the sponge-derived Pseudoalteromonas sp. PA2MD11.

Active heterotrophic metabolism is a critical metabolic role performed by sponge-associated microorganisms, but little is known about their capacity to metabolize marine polysaccharides (MPs). Here, we investigated the genome of the sponge-derived Pseudoalteromonas sp. strain PA2MD11 focusing on its macroalgal carbohydrate-degrading potential. Carbohydrate-active enzymes (CAZymes) for the depolymerization of agar and alginate were found in PA2MD11's genome, including glycoside hydrolases (GHs) and polysaccharide lyases (PLs) belonging to families GH16, GH50 and GH117, and PL6 and PL17, respectively. A gene potentially encoding a sulfatase was also identified, which may play a role in the strain's ability to consume carrageenans. The complete metabolism of agar and alginate by PA2MD11 could also be predicted and was consistent with the results obtained in physiological assays. The polysaccharide utilization locus (PUL) potentially involved in the metabolism of agarose contained mobile genetic elements from other marine Gammaproteobacteria and its unusual larger size might be due to gene duplication events. Homology modelling and structural protein analyses of the agarases, alginate lyases and sulfatase depicted clear conservation of catalytic machinery and protein folding together with suitable industrially-relevant features. Pseudoalteromonas sp. PA2MD11 is therefore a source of potential MP-degrading biocatalysts for biorefinery applications and in the preparation of pharmacologically-active oligosaccharides.

Discovery of a fucoidan endo-4O-sulfatase: Regioselective 4O-desulfation of fucoidans and its effect on anticancer activity in vitro.

Fucoidans are a class of sulfated fucose-containing bioactive polysaccharides produced by brown algae. The biological effects exhibited by fucoidans are thought to be related to their sulfation. However, the lack of methods for sulfation control does not allow for a reliable conclusion about the influence of the position of certain sulfate groups on the observed biological effects. We identified the gene encoding the endo-acting fucoidan sulfatase swf5 in the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. This is the first report on the sequence of fucoidan endo-sulfatase. Sulfatase SWF5 belongs to the subfamily S1_22 of the family S1. SWF5 was shown to remove 4O-sulfation in fucoidans composed from the alternating α-(1→3)- and α-(1→4)-linked residues of sulfated L-fucose but not from fucoidans with the α-(1→3)-linked backbone. The endo-sulfatase was used to selectively prepare 4O-desulfated fucoidan derivatives. It was shown that the 4O-desulfated fucoidans inhibit colony formation of DLD-1 and MCF-7 cells less effectively than unmodified fucoidans. Presumably, 4O-sulfation makes a significant contribution to the anticancer activity of fucoidans.

Post-Translational Formation of Aminomalonate by a Promiscuous Peptide-Modifying Radical SAM Enzyme.

Aminomalonate (Ama) is a widespread structural motif in Nature, whereas its biosynthetic route is only partially understood. In this study, we show that a radical S-adenosylmethionine (rSAM) enzyme involved in cyclophane biosynthesis exhibits remarkable catalytic promiscuity. This enzyme, named three-residue cyclophane forming enzyme (3-CyFE), mainly produces cyclophane in vivo, whereas it produces formylglycine (FGly) as a major product and barely produce cyclophane in vitro. Importantly, the enzyme can further oxidize FGly to produce Ama. Bioinformatic study revealed that 3-CyFEs have evolved from a common ancestor with anaerobic sulfatase maturases (anSMEs), and possess a similar set of catalytic residues with anSMEs. Remarkably, the enzyme does not need leader peptide for activity and is fully active on a truncated peptide containing only 5 amino acids of the core sequence. Our work discloses the first ribosomal path towards Ama formation, providing a possible hint for the rich occurrence of Ama in Nature.

New Protocol-Guided Exploitation of a Lysosomal Sulfatase Inhibitor to Suppress Cell Growth in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a highly invasive and aggressive malignant glioma. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma, so more effective strategies of antitumor treatments are in urgent demand. Here, we found that lysosomal sulfatase expression was significantly correlated with poor prognosis of GBM. Hence, a new probe, MNG, was developed with a new protocol utilizing glucose groups to detect lysosomal sulfatase. It also exhibits potential for monitoring GBM cells, depending on the hyperactive lysosomal sulfatase expression of tumor cells. Meantime, we identified that sulbactam as the first reported lysosomal sulfatase inhibitor inhibits the tumor growth of GBM. Collectively, our work highlights that lysosomal sulfatase was detected using a new protocol and its potential as a therapeutic target in GBM and reveals a distinct mechanism that sulbactam inhibits cell proliferation related to lysosomal sulfatase, indicating that sulbactam could be a promising therapeutic agent against GBM.

Self-buffering capacity of a human sulfatase for central nervous system delivery.

Direct delivery of therapeutic enzymes to the Central Nervous System requires stringent formulation design. Not only should the formulation design consider the delicate balance of existing ions, proteins, and osmolality in the cerebrospinal fluid, it must also provide long term efficacy and stability for the enzyme. One fundamental approach to this predicament is designing formulations with no buffering species. In this study, we report a high concentration, saline-based formulation for a human sulfatase for its delivery into the intrathecal space. A high concentration formulation (≤ 40 mg/mL) was developed through a series of systematic studies that demonstrated the feasibility of a self-buffered formulation for this molecule. The self-buffering capacity phenomenon was found to be a product of both the protein itself and potentially the residual phosphates associated with the protein. To date, the self-buffered formulation for this molecule has been stable for up to 4 years when stored at 5 ± 3 °C, with no changes either in the pH values or other quality attributes of the molecule. The high concentration self-buffered protein formulation was also observed to be stable when exposed to multiple freeze-thaw cycles and was robust during in-use and agitation studies.

Purification, Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis.

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.

Investigation of action pattern of a novel chondroitin sulfate/dermatan sulfate 4-O-endosulfatase.

Recently, a novel CS/DS 4-O-endosulfatase was identified from a marine bacterium and its catalytic mechanism was investigated further (Wang, W., et. al (2015) J. Biol. Chem.290, 7823-7832; Wang, S., et. al (2019) Front. Microbiol.10, 1309). In the study herein, we provide new insight about the structural characteristics of the substrate which determine the activity of this enzyme. The substrate specificities of the 4-O-endosulfatase were probed by using libraries of structure-defined CS/DS oligosaccharides issued from synthetic and enzymatic sources. We found that this 4-O-endosulfatase effectively remove the 4-O-sulfate of disaccharide sequences GlcUAß1-3GalNAc(4S) or GlcUAß1-3GalNAc(4S,6S) in all tested hexasaccharides. The sulfated GalNac residue is resistant to the enzyme when adjacent uronic residues are sulfated as shown by the lack of enzymatic desulfation of GlcUAß1-3GalNAc(4S) connected to a disaccharide GlcUA(2S)ß1-3GalNAc(6S) in an octasaccharide. The 3-O-sulfation of GlcUA was also shown to hinder the action of this enzyme. The 4-O-endosulfatase exhibited an oriented action from the reducing to the non-reducing whatever the saturation or not of the non-reducing end. Finally, the activity of the 4-O-endosulfatase decreases with the increase in substrate size. With the deeper understanding of this novel 4-O-endosulfatase, such chondroitin sulfate (CS)/dermatan sulfate (DS) sulfatase is a useful tool for exploring the structure-function relationship of CS/DS.

Chemical Tools for Illumination of Tuberculosis Biology, Virulence Mechanisms, and Diagnosis.

Tuberculosis (TB) remains one of the deadliest infectious diseases and begs the scientific community to up the ante for research and exploration of completely novel therapeutic avenues. Chemical biology-inspired design of tunable chemical tools has aided in clinical diagnosis, facilitated discovery of therapeutics, and begun to enable investigation of virulence mechanisms at the host-pathogen interface of Mycobacterium tuberculosis. This Perspective highlights chemical tools specific to mycobacterial proteins and the cell lipid envelope that have furnished rapid and selective diagnostic strategies and provided unprecedented insights into the function of the mycobacterial proteome and lipidome. We discuss chemical tools that have enabled elucidating otherwise intractable biological processes by leveraging the unique lipid and metabolite repertoire of mycobacterial species. Some of these probes represent exciting starting points with the potential to illuminate poorly understood aspects of mycobacterial pathogenesis, particularly the host membrane-pathogen interactions.

Lysosomal sulfatases: a growing family.

Sulfatases constitute a family of enzymes that specifically act in the hydrolytic degradation of sulfated metabolites by removing sulfate monoesters from various substrates, particularly glycolipids and glycosaminoglycans. A common essential feature of all known eukaryotic sulfatases is the posttranslational modification of a critical cysteine residue in their active site by oxidation to formylglycine (FGly), which is mediated by the FGly-generating enzyme in the endoplasmic reticulum and is indispensable for catalytic activity. The majority of the so far described sulfatases localize intracellularly to lysosomes, where they act in different catabolic pathways. Mutations in genes coding for lysosomal sulfatases lead to an accumulation of the sulfated substrates in lysosomes, resulting in impaired cellular function and multisystemic disorders presenting as lysosomal storage diseases, which also cover the mucopolysaccharidoses and metachromatic leukodystrophy. Bioinformatics analysis of the eukaryotic genomes revealed, besides the well described and long known disease-associated sulfatases, additional genes coding for putative enzymes with sulfatases activity, including arylsulfatase G as well as the arylsulfatases H, I, J and K, respectively. In this article, we review current knowledge about lysosomal sulfatases with a special focus on the just recently characterized family members arylsulfatase G and arylsulfatase K.

Synthesis of metronidazole based thiazolidinone analogs as promising antiamoebic agents.

Metronidazole and its derivatives are widely used for the treatment of amoebiasis. However, metronidazole is considered as the standard drug but it has many side effects. The present study describes the synthesis of a series of metronidazole based thiazolidinone analogs via Knoevenagel condensation of 4-[2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethoxy]benzaldehyde 1 with various thiazolidinone derivatives 2-14 to get the new scaffold (15-27) having better activity and lesser toxicity. Six compounds have shown better efficacy and lesser cytotoxicity than the standard drug metronidazole towards HM1: IMSS strain of Entamoeba histolytica. These compounds may combat the problem of drug resistance and might be effective in identifying potential alternatives for future drug discovery against EhOASS.

Structural Insights into Endobiotic Reactivation by Human Gut Microbiome-Encoded Sulfatases.

Phase II drug metabolism inactivates xenobiotics and endobiotics through the addition of either a glucuronic acid or sulfate moiety prior to excretion, often via the gastrointestinal tract. While the human gut microbial ß-glucuronidase enzymes that reactivate glucuronide conjugates in the intestines are becoming well characterized and even controlled by targeted inhibitors, the sulfatases encoded by the human gut microbiome have not been comprehensively examined. Gut microbial sulfatases are poised to reactivate xenobiotics and endobiotics, which are then capable of undergoing enterohepatic recirculation or exerting local effects on the gut epithelium. Here, using protein structure-guided methods, we identify 728 distinct microbiome-encoded sulfatase proteins from the 4.8 million unique proteins present in the Human Microbiome Project Stool Sample database and 1766 gut microbial sulfatases from the 9.9 million sequences in the Integrated Gene Catalogue. We purify a representative set of these sulfatases, elucidate crystal structures, and pinpoint unique structural motifs essential to endobiotic sulfate processing. Gut microbial sulfatases differentially process sulfated forms of the neurotransmitters serotonin and dopamine, and the hormones melatonin, estrone, dehydroepiandrosterone, and thyroxine in a manner dependent both on variabilities in active site architecture and on markedly distinct oligomeric states. Taken together, these data provide initial insights into the structural and functional diversity of gut microbial sulfatases, providing a path toward defining the roles these enzymes play in health and disease.